The term "binding pair" refers to two molecules that bind each other with high affinity. Many binding pair systems presently exist for the purification and targeting of tagged compounds. The most widely-used system at present is the biotin-streptavidin binding pair that has also been applied to in vivo tumour targeting methods (Paganelli et al., 1991). In addition, recombinant fusion proteins expressing the myc or Flag tags have been isolated and purified using antibodies directed against these tags (Munro and Pelham, 1986; Hopp et al., 1988). Poly-his peptide tails have been genetically fused to recombinant proteins and purified on nickel-coated agarose or using affinity columns (Skerra et al., 1991). The streptavidin-binding strep tag (Schmidt and Skerra, 1993) has been used in similar applications.
However, the application of each of the above examples is restricted. Techniques such as affinity purification require a specific yet low affinity interaction so as not to impair the function of the recombinant protein with a harsh elution protocol. Conversely, targeting and specific immobilization both require high affinity binding of the tag with a long half-life of interaction of the binding pair. The systems described in the prior art that employ binding pairs do not possess this flexibility, and thus are not universally applicable.
Calmodulin is a calcium dependent binding protein that regulates a wide range of enzymes, and plays a key role in intracellular signal transduction. It is also one of the few examples of a small protein capable of binding ligands with a high affinity.
Due to their small size, calmodulin-ligand compelexes have been used as convenient models for the study of protein association and dissociation. Since the elucidation of three-dimensional structures of calmodulin in complex with high affinity ligand substrates, this system has been amenable to the study of the mechanisms by which proteins recognise one another in high affinity interactions.
Pending patent application WO95/12672 discloses the use of binding pairs comprising calmodulin and calmodulin ligands for the detection, immobilization, targeting and purification of recombinant polypeptides. This system is attractive since the binding interaction can be modified through changes in calcium concentration that can easily be controlled through the use of calcium chelators. This greatly broadens the applicability of binding pairs as experimental and therapeutic tools.
Stoko-Hahn et al., (1992) have made a fusion of a calmodulin binding ligand tag derived from the C terminus of rabbit skeletal muscle myosin light chain kinase (sMLCK), and a recombinant protein. They have used this in purification strategies on an affinity support. In the presence of high levels of calcium, this system displays a specific high affinity interaction, the dissociation constant for the binding pair being of the order of 3 nM. The addition of EGTA (a calcium chelator) lowers the affinity of the interaction, allowing a very mild elution protocol, and meaning that denaturation or disruption of the native protein structure is unlikely. However, although the affinity of calmodulin towards calmodulin ligands is high, it is not sufficiently high for many targeting, immobilisation and particularly therapeutic applications.
For many therapeutic applications, sub nanomolar or picomolar dissociation constants, high association rate constants and extremely low dissociation rate constants are necessary to ensure efficient targeting of the complex and a sufficiently long half-life of the binding interaction. The biotin-streptavidin binding pair is widely used both experimentally and therapeutically for the targeting, purification and immobilization of proteins. The main attraction of this system lies in the high affinity of interaction (K.sub.d =10.sup.-15 M) of the binding pair.
However, a significant disadvantage of using this binding pair is the immunogenicity of streptavidin in the human body. This greatly limits the utility of this system for therapeutic applications. There is thus a great need for an experimentally malleable binding pair system that possesses both a high affinity interaction and low immunogenicity.